Process for making (2S,5S)-5-fluoromethylornithine

ABSTRACT

PCT No. PCT/US93/11283 Sec. 371 Date Jul. 18, 1995 Sec. 102(e) Date Jul. 18, 1995 PCT Filed Nov. 19, 1993 PCT Pub. No. WO94/17795 PCT Pub. Date Aug. 18, 1994The present invention is directed to a process for making (2S,5S)-5-fluoromethylornithine through the reaction of wet penicillin acylase with (2R,5R) and (2S,5S)-6-fluoromethyl-3-(phenylactyl)-amino-2-piperidone and the addition of an acid to the resulting product.

This application is a 371 of PCT/US93/11283 filed Nov. 19, 1993.

The present invention comprises the use of certain inhibitors ofornithine aminotransferase, and more preferably, certain 5-substitutedornithine derivatives for the treatment of dementia of the Alzheimer'stype (DAT) alone and in combination with other agents.

BACKGROUND OF THE INVENTION

The ammonium ion (NH₄ +) serves a major role in the maintenance ofacid-base balance, but is toxic in high concentrations. By the body itis produced from many precursors (nucleic acids, proteins, amino acids,hexosamines, primary amines) by different reactions, and introduced intothe body by exogenous sources such as the break down of dietary proteinsby intestinal bacteria.

About 20% of the urea [CO(NH₂)₂ ] produced in the body diffuses into thegut where it is converted by bacteria to ammonia and carbon dioxide. Theammonia is absorbed and converted back to urea in the liver by way ofthe ornithine (urea) cycle, the major pathway for elimination ofammonia. Thus, acute and chronic diseases of the liver impair theability of the liver to remove ammonia from the body.

Elevated levels of ammonia can easily pass the blood brain barriercausing encephalopathies (degenerative diseases of the brain). One causeof neurogenic encephalopathy is bacterial infections of the urinarytract (e.g. the neurogenic bladder). Another cause is deficientdetoxification of ammonia due to acute or chronic liver disease whichleads to hepatogenic encephalopathy. An important factor in thepathogenesis of this disease has been identified as exogenous(gastrointestinal) ammonia.

Proteins, nucleic acids, amino acids and hexosamines have long beensuggested as other sources of cerebral ammonia. Oxidative deaminationsof primary amines (monoamines, diamines and polyamines), glycinecatabolism via the glycine cleavage system, deaminations of purines andpyrimidines and glucosamine-6-phosphate, among others, are well knownammonia generating reactions, which may contribute to the steady-statelevel of brain ammonia.

Dementia of the Alzheimer's Type (DAT) is another type of degenerativebrain disease with unknown etiology (although several hypotheses havebeen postulated). There have been recent reports of not only elevatedconcentrations of ammonia in the brain of DAT patients, but also reportsthat ammonia is endogenously generated in excess therein. Hoyer, S., etal., Neurosci. Lett. 117:358-362 (1990). There were two reports thatarterial ammonia levels were significantly higher in DAT patients thanin appropriately matched control subjects. Fisman, M., et al., Am. J.Psychiatry 142:71-73 (1985); Fisman, M., et al., J. Am. Ger. Soc.37:1102 (1989). Patients who met the diagnostic criteria of DAT, but hadno liver disease, nor urinary tract infections had levels of 208±136 μgammonia per 100 ml of plasma. The normal range was 20-94 μg/100 ml; 83%of the patients had blood ammonia concentrations above the normallimits. Branconnier, R. J., et al., Am. J. Psychiatry 143: 1313 (1986).Arterio-venous differences of ammonia in patients suffering fromadvanced DAT, and in patients clinically diagnosed as having incipientdementia, in all probability DAT of early onset, were reported. Healthyvolunteers showed an average ammonia uptake by the brain of 72±7μg.kg⁻¹.min.⁻¹. In striking contrast, 27±3 μg.kg⁻¹. min.⁻¹ of ammoniawas released from the brains of patients with advanced DAT. Patientswith presumed early-onset DAT released 256±162 μg.kg⁻¹ .min.⁻¹ ammoniainto the circulation. These findings suggest excessive ammoniaproduction within the brain, with or without a deficient mechanism ofammonia detoxification. Hoyer, S., et al. Neurosci. Lett. 117:358-368(1990).

The present invention recognizes hyperammonemia as an important factorin at least the symptomatology and progression of DAT. As furtherdescribed hereafter, cerebral hyperammonemia may influence those factorswhich are considered to be hallmarks of DAT.

CEREBRAL HYPERAMMONEMIA AND DAT

a) SYNAPTIC TRANSMISSION IN AMMONIA INTOXICATION

Ammonia is capable of interfering with the function of the majorexcitatory (glutamatergic) and the major inhibitory (GABAergic) neuronalsystems of the vertebrate central nervous system which is impaired inthe patient having DAT.

Based on experimental results it was calculated that an increase ofammonia to about 0.5 μmol.g⁻¹ brain i.e. a 2-5-fold increase, issufficient to disturb excitatory and inhibitory synaptic transmissionand to initiate the encephalopathy related to acute ammonia intoxicationRaabe, W., Neurochem. Pathol. 6: 145-166 (1987). Thus, it seems evidentthat slowly progressing pathogenic mechanisms may be initiated even atbrain ammonia concentrations only slightly above physiological levels.

Glutamate-mediated excitatory synaptic transmission is decreased byammonia. Whether this effect is related to a depletion of glutamate inpresynaptic terminals is unclear at present.

Inhibitory synaptic transmission is also decreased by ammonia, byhyperpolarizing Cl⁻ -dependent inhibitory (e.g. GABAergic) neurons. Thiseffect is related to the inactivation of the extrusion of Cl⁻ fromneurons by ammonia. By the same action ammonia also decreases thehyperpolarizing action of Ca²⁺ - and voltage dependent Cl⁻ -currents.Since a large proportion of the GABAergic and other inhibitory neuronscontrol inhibitory inputs, ammonia produces an increase in neuronalexcitation by "disinhibition".

b) REDUCED GLUCOSE UTILIZATION

Most conspicuous findings of experimental and human diseases withhyperammonemic states, namely the impairment of brain glucoseutilization, with concomitantly decreased rates of energy metabolism andastrocytic alterations, characterized as "Alzheimer type II gliosis" arecharacteristic for DAT brains as well: in PET (positron emissiontomography) studies cerebral glucose utilization was found to bepredominantly reduced in the parieto-temporal cortex. Overall cerebralglucose utilization was found to be diminished by about 50% with normaloxygen consumption in early-onset, but reduced oxygen consumption inlate onset DAT. The impairment of brain energy metabolism in DAT, and ofenzymes involved in energy metabolism, has subsequently been reported byseveral investigators.

c) INTERFERENCE WITH GLIA FUNCTION

Astrocyte abnormalities are a characteristic of DAT. Observationssupporting the idea that reactive astrocytes may mediate neuropathologicevents of DAT, including the facilitation of extracellular depositionsof β-amyloid protein have been reported. Frederickson, Neurobiol. Aging13:239-253 (1992).

Astrocytic damage by ammonia is followed by a decrease of glutaminesynthetase activity, as was evidenced from the reduction of the activityof this enzyme by 15% in rats with portacaval shunts, Butterworth, R.F., et al., J. Neurochem. 51:486-490 (1988). However, this decrease insynthetase activity may cause further damage to astrocytes. It is wellestablished that glutamine synthetase is critically involved in theregulation of intracellular ammonia and acid-base balance. Anyderangement of the function of this enzyme will be followed by theamplification of ammonia toxicity. Therefore, it is not surprising thatan increased intracellular pH, and swelling of astrocytes was observedin hyperammonemic rats, Swain, M. S., et al. Am. J. Physiol.261:R1491-51496 (1991).

Increasing evidence emerges for a role of microglia in DAT pathologyMcGeer, P. L., et al. Can. J. Neurol. Sci. 18: 376-379 (1991). Thesecells are seen in many degenerating cells, and virtually every senileplaque has microglial cells or cell processes in the plaque. It isbelieved that microglia invasion is an indication for the brain'sattempt to rid itself of cell debris. Since β-amyloid precursor proteinis likely to be formed in microglia these cells may contribute to theformation of β-amyloid protein depositions in two ways, by phagocytosisof nerve ending membranes, and by their intrinsic β-amyloid precursorprotein.

d) HYPERAMMONEMIA AND EXCITOTOXIC AMINO ACIDS

Presumably the most conspicuous difference between the amino acidpatterns of cirrhotic and DAT patients is the several-fold increase ofglutamine in all brain regions of cirrhotics, but no change in theconcentration of this amino acid in the brains of DAT patients.Likewise, no increase of glutamine was detected in the cerebrospinalfluid (CSF) of patients with DAT, whereas the levels of this amino acidwere elevated in the CSF of experimental animals with portal-systemicencephalopathy. These findings suggest the inability of the brains ofDAT patients to enhance glutamine formation above a certain level andmay be taken as an indication for a considerable sensitivity of DATbrains even to small increases in the rate of ammonia formation. Due tothe elevation of ammonia levels, reductive amination of 2-oxoglutarate(catalyzed by glutamate dehydrogenase) may take place, both in DAT andhepatogenic encephalopathy. Presumably, this "extra" glutamate can onlybe removed from the brain as glutamine in the latter disease not in DATbrains, due to its limited glutamine synthetase activity. Glutamateformation from 2-oxoglutarate impairs at the same time energymetabolism, by decreasing the equilibrium concentration of thissubstrate of the tricarboxylic acid cycle.

Glutamate concentrations are lower in the brains of DAT patients than inage-matched controls, due to losses of glutamatergic neurons, but CSFlevels of glutamate are elevated, both in DAT, Pomara, N., et al. Am. J.Psychiatry 149: 251-254 (1992), and in portal systemic encephalopathyTherrien, G., et al., Metabolic Brain Dis. 6:65-74 (1991), indicatingenhanced extracellular concentrations of this amino acid. Disregardingthe mentioned possibility of the enhanced formation of glutamate byreductive amination of 2-oxoglutarate the increase of extracellularglutamate concentrations is most probably a result of the impairment ofthe uptake of glutamate into perineuronal astrocytes due to the derangedastrocyte function by ammonia. Since it is well established that theneurotoxic effects of glutamate are enhanced by inhibition of uptakesites, derangement of glial uptake mechanisms could be a major reasonfor excitotoxic cell damage in DAT.

The release of aspartate from the brains of patients with early-onsetDAT is indicative for a further cause of excitotoxic damage during acertain stage of the disease. Patients with a mean age of 60 years hadnormal CSF levels of aspartate. Pomara, N., et al., Am. J. Psychiatry149: 251-254 (1992).

There is evidence for the selective loss of glutamate receptors incortex and hippocampus of DAT brains. In the cerebellum ofhyperammonemic rats a decrease of the number of both high- andlow-affinity binding sites of glutamate was noticed. The decrease wasonly in the N-methyl-D-aspartate-specific binding sites, without anyalterations in the binding sites of kainate or quisqualate. Theseeffects were mimicked when the membrane preparations from normal animalswere incubated with ammonium acetate. Binding of muscimol (a GABAreceptor agonist) was enhanced under the same experimental conditionsRaghavendra Rao, V. L., et al. Neurosci. Lett. 130:251-254 (1991). Theseobservations show again the ability of ammonia to affect functions ofboth glutamatergic and GABAergic neurons.

The compounds of the present invention have been described in EuropeanPatent application number 88400275.9 filed Feb. 5, 1988, publicationnumber 0 326 766 entitled 5-Substituted Ornithine Derivatives, which ishereby incorporated herein. These compounds were disclosed therein to beeffective in treating conditional deficiencies of ornithine and in casesof ammonia intoxication.

Ornithine is a substrate in the urea cycle. The urea cycle is effectivein incorporating ammonium ions into urea in order to be eliminated fromthe body. The compounds of the present invention inactivate ornithine:2-oxoacid aminotransferase (OAT). It is believed that by enhancing thelevel of tissue ornithine concentrations due to inactivation of OAT overan extended period of time, urea formation in the liver and presumablyin some other tissues would be a consequence thereof, thereby loweringblood and cerebrospinal fluid ammonia concentrations. These compoundsare useful in numerous well known human illnesses associated withelevated blood and cerebrospinal fluid ammonia concentrations, amongwhich, for example, are liver cirrhosis, fulminant hepatic failure andurinary tract/bladder infections.

Despite the foregoing, DAT has not previously been thought of as acondition benefited by the lowering of ammonia levels. Indeed, the useof OAT inhibitors presents an especially effective method of treatmentfor DAT since most Alzheimer patients usually have normal liverfunction. Thus the use of the compounds of the present invention presenta much needed new approach to the treatment of DAT.

It is an object of the present invention to provide a new use for thecompounds of the present invention by treating DAT. It is another objectof the present invention to provide a synthesis for an enantiomer of thecompounds of the present invention and yet another objective is topresent combination therapy useful in the treatment of DAT.

SUMMARY OF THE PRESENT INVENTION

The present invention comprises the new use for the treatment of DATwith OAT inactivators, preferably substituted ornithine derivatives andmore preferably 5-substituted ornithine derivatives of the formula:##STR1## wherein R is a --CH₂ F, CHF₂, --CHClF, --C.tbd.CH, CH═CH₂ orCH═C═CH₂ group, stereoisomer, or a pharmaceutically acceptable acidaddition salt thereof. The treatment of DAT may be a combination therapycomprising administration of OAT inhibitors and other agents useful inlowering brain ammonia levels in the patient.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

As used herein "DAT" or "dementia of the Alzheimer's type" meansprogressively deteriorating organic mental syndromes in which there isimpairment in short-term and long-term memory. This degenerativedementia can be mild (impairment of work or social activities but ableto live alone), moderate (some degree of supervision needed) or severe(continual supervision required).

Impairment in short-term memory is the inability to learn newinformation and may be demonstrated by, for example, the patients'inability to remember three objects after five minutes. Long-term memoryimpairment is the inability to remember information that was known inthe past and may be indicated by, for example, the patients' inabilityto remember past personal information such as their birthplace,occupation, what happened yesterday, etc., or the inability to rememberfacts of common knowledge. There is typically impairment in abstractthinking, impairment in judgment, personality changes or otherdisturbances of higher cortical functions.

"Patient" as used herein means warm-blooded animals, such as for examplerats, mice, dogs, cats, guinea pigs, primates and humans. The term"treat", or forms thereof, means to prevent or alleviate the patient'sdisease or condition.

The term "stereoisomer" is a general term for all isomers of individualmolecules that differ only in the orientation of their atoms in space.It includes mirror image isomers (enantiomers), geometric (cis/trans)isomers, and isomers of compound with more than one chiral center thatare not mirror images of one another (diastereoisomers). In the presentinvention, racemic mixtures of compounds of formula I may have fourenantiomers and only one of the four enantiomers may be preferred overthe others.

The term "combination therapy" can mean concurrent or consecutiveadministration of two or more agents. For example, concurrentadministration can mean one dosage form in which the two or more agentsare contained whereas consecutive administration can mean separatedosage forms administered to the patient at different times and maybeeven by different routes of administration.

Any agent may be used in combination therapy with the compounds of thepresent invention which are useful in treating DAT. Preferably agentsuseful in lowering ammonia levels in the brain of DAT patients are used.For example, agents useful in promoting the excretion of ammonia throughthe urea cycle such as ornithine, citrulline and arginine may be used.Preferably, agents which are believed to lower brain ammonia levelsindependent of the urea cycle are used such as L-acetylcarnitine andL-carnitine.

As in most inventions, there are preferred embodiments. In the presentinvention it is preferred that R is CH₂ F (Example 1 in EP 326 766), andmost preferably, one of its enantiomers which is believed to be(S/S)-2,5-diamino-6-fluoro-hexanoic acid.

EXAMPLE 1

2,5-Diamino-6-fluoro-hexanoic acid is obtained as described in Europeanpatent 326 766. The enantiomers are separated using any appropriatemethod such as HPLC, or alternative methods may be used as follows,##STR2##

(2R, 5R) and (2S,5S) -Methyl-3-amino-2-piperidone-6-carboxylate

2,5-Diazabicyclo (2.2.2) octane-3,6-dione [J. Med. Chem. 1974, 17,481-487, P. A. Sturm, D. W. Henry, Antifilarial agents.Diazabicyclooctanes and Diazabicycloheptanes as bridged analogs ofdiethyl carbamazine] (10.75 g, 0.0768 mol) is dissolved in a solution ofhydrochloric acid in anhydrous methyl alcohol (560 ml, 0.27 M). Themixture is stirred at 20° C. for 18 hours, then neutralized with silvercarbonate (21.5 g, 0.082 mol) and filtered over a celite pad. Thesolvent is evaporated and the residue is dried overnight. The titlecompound is obtained as a white solid (12.8 g, 97% yield).

¹ H NMR (360 MHz, CD₃ OD) δ ppm: 3.85 (m, 1H, CHCOO); 3.55 ,(m, 1H,CHNH₂); 3.40 (s, 3H, CH₃); 1.9 and 1.4 (2m, 2×2H CH₂ CH₂).

MS: monoTFA derivatives: m/e=269 (MH⁺); 286 (MNH₄ ⁺) Chiral GC: 145° C.,1 bar H₂, TFA derivatives. 2 peaks: 15.63 min and 16.64 min.

(2R, 5R) and(2S,5S)-Methyl-3-(phenylacetyl)amino-2-piperidone-6-carboxylate

To a mixture of phenylacetic acid (10.2 g, 0.075 mol), dicyclohexylcarbodiimide (13.5 g, 0.075 mol), pyridine (7.2 ml, 0.061 mol) and4-dimethylaminopyridine (0.9 g, 0.0073 mol) in anhydrous dichloromethane(400 ml), is added (2R,5R) and(2S,5S)-methyl-3-amino-2-piperidone-6-carboxylate (12.8 g, 0.0744 mol).The reaction mixture is stirred at 20° C. for 24 hours, then thedicyclohexylurea formed is filtered off and the filtrate is evaporated.The residue is purified by flash chromatography on silica using methylalcohol/ethyl acetate 5/95 as eluent. The title compound is obtained asa white solid (19.61 g, 93% yield).

¹ H NMR (200 MHz, CD₃ OD) δ ppm: 7.87 (m, 5H, Ph); 6.55 (d, 1H, NH);6.40 (s, 1H, NH-1); 4.35 (m, 1H, H-3); 4.12 (m, 1H, H-6); 3.77 (s, 3H,COOMe); 3.54 (s, 2H, COCH₂ Ph); 2.5 (m, 1H, H-4A); 2.2 (m, 2H, H-5); 1.5(m, 1H, H-4B).

HPLC: Chiralpak AD, 250×4.6 mm, 21° C., EtOH/MeOH/heptane: 25/45/30, 1ml/min, 210 nm, 2 peaks: 7.26 min and 12.51 min.

Analysis calculated for C₁₅ H₁₈ N₂ O₄ (290.31): C, 62.06; H, 6.25; N,9.65. Found: C, 62.50; H, 6.26; N, 9.73. m.p. 138° C.

(2R,5R) and (2S,5S)-3-(Phenylacetyl)amino-2-piperidone-6-methylalcohol

To a slurry of (2R,5R) and(2S,5S)-methyl-3-(phenylacetyl)amino-2-piperidone-6-carboxylate (1.451g, 0.005 mol) in anhydrous diethyl ether (50 ml) and tetrahydrofuran(12.5 ml) is added lithium borohydride (0.109 ml, 0.005 mol) andlithium-tri-sec-butylborohydride (0.5 ml, 1M, 0.0005 mol). The reactionmixture is refluxed for 12 hours, then methyl alcohol is added (5 ml)and the solvents are evaporated. The residue is purified by flashchromatography on silica gel using methyl alcohol/ethyl acetate (8/92)as eluent. After recrystallization, the title compound is obtained as awhite solid (2.275 g, 97% yield).

¹ H NMR (200 MHz, CD₃ OD) δ ppm: 7.7 (m, 5H, ph); 4.05 (m, 1H, H-3);3.25 (m, 3H, CHCH₂ O); 1.55 (m, 4H, H-4, H-5).

HPLC: Chiralpak AD, 250×4.6 mm, 21° C., EtOH/heptane: 60/40, 1ml/min,210 nm 2 peaks: 5.37 min and 6.48 min.

Analysis calculated for C₁₄ H₁₈ N₂ O₂ (262.311): C, 64.11; H, 6.92; N,10.68. Found: C, 64.12; H, 6.86; N, 10.68. m.p. 158° C.

(2R,5R) and (2S ,5S)-6-Fluoromethyl-3-(phenylacetyl)amino-2-piperidone

A suspension of (2R,5R) and(2S,5S)-3-(phenylacetyl)-amino-2-piperidone-6-methylalcohol (52.4 mg,0.2 mmol) in 5 ml anhydrous dichloromethane is cooled to -78° C.Dimethylaminosulfurtrifluoride (53.2 mg, 0.4 ml, 0.4 mmol) is addedslowly to the mixture. After 5 minutes at -78° C., the reaction mixtureis allowed to reach room temperature, and stirred for another 16 hours.The reaction is quenched with icy water. The organic phase is dilutedwith dichloromethane (25 ml) and washed with water. The organic phase isdried over sodium sulfate, filtered and the solvents are evaporated. Theresidue is purified by flash chromatography on neutral aluminum oxydeactivity III using methyl alcohol/ethyl acetate: 8/92 as eluent. Afterrecrystallization in chloroform/pentane the title compound is obtainedas white crystals (0.024 g, 45% yield).

¹ H NMR (360 MHz, CD₃ OD) δ ppm: 7.3 (m, 5H, C₆ H₅); 6.47 (d, 1H,NHCOCH₂ Ph); 6.27 (s, 1H, NH); 4.35 (dAB, H_(A), JH_(A) F=46.4 Hz) 4.33(dAB, 1H, JH_(B) F=47.33 Hz); 4.24 (m, 1H, H-3); 3.75 (m, 1H, H-6); 3.6(s, 2H, CH₂ Ph); 2.42 (m, 1H, H₂).

¹⁹ F NMR (338.8 MHz, CHCl₃) δ ppm: -62.82 (dt, JHF=46.8 Hz). HPLCChiralpak AD, 250×4.6 mm, 21° C., EtOH/heptane: 60/40, 0.5 ml/min, 210nm. 2 peaks: 12.8 min and 14.8 min.

Analysis calculated for C₁₄ H₁₇ N₂ O₂ F (264.30): C, 63.62; H, 6.48; N,10.60. Found: C, 63.17; H, 6.56; N, 10.52.

(2R, 5R) and (2S,5S)-6-Fluoromethyl-3-(phenylacetyl)amino-2-piperidoneand (2S,5S)-6-Fluoromethyl-3-amino-2-piperidone

To a solution of (2R,5R) and(2S,5S)-6-fluoromethyl-3-(phenylacetyl)-amino-2-piperidone (0.097 g,0.37 mmol) in phosphate buffer pH 7.0 (11 ml, 0.1M) is added wetpenicillin acylase (0.040 g). After stirring for 30 minutes, the enzymeis filtered out, the solution is washed with dichloromethane to removethe (2R,5R)-6-fluoromethyl-3-(phenylacetyl)-amino-2-piperidone (0.057g). Evaporation of the aqueous phase affords the(2S,5S)-6-fluoromethyl-3-amino-2-piperidone as a white solid (0.023 g).

HPLC: Chiralpak AD, 250×4.6 mm, 21° C., EtOH/heptane: 60/40; 0.5 ml/min,210 nm. 1 peak: 14.8 min.

(2S,5S)-6-Fluoro-2,5-diaminohexanoic acid, dihydrochloride, and((2S,5S)-5-fluoromethylornithine, dihydrochloride)

A solution of (2S,5S)-6-fluoromethyl-3-amino-2-piperidone (0.020 g, 0.17mmol) in hydrochloric acid (1 ml, 6N) is refluxed for 2.5 hours. Thesolution is diluted with water (2 ml) and washed 4 times withdichloromethane (4 ml), the aqueous phase is evaporated and the residueis recrystallized in methyl alcohol/diethyl ether. The title compound isobtained as white crystals (15 mg, 82% yield).

¹ H NMR (360 MHz, CD₃ OD) δ ppm: 4.4 (m, 2H, CH₂ F); 3.8 (m, 1H, H-2);3.3 (m, 1H, H-5); 1.7 (m, 4H, CH₂ CH₂).

¹⁹ F NMR (338.8 MHz, CHCl₃) δ ppm: -69.15 (dt, ³ J=23 Hz, ² J=47 Hz).

EXAMPLE 2

In order to test the efficacy of treatment with the compounds of thepresent invention, blood ammonia concentration can be measured in venousblood, by using an ammonia specific electrode according to the method ofH. F. Proelss, et al., Clin. Chem. 19: 1162-1169 (1973), incorporatedherein by reference. In order to minimize liberation of bound ammonia byhydrolytic processes the blood samples will be cooled immediately to 0°C. and deproteinized by mixing with an equal volume of 0.4M perchloricacid. After dilution 1:1 of the mixture with 0.2M perchloric acid, theproteins will be removed by centrifugation. Ammonia concentrations inthe clear supernatants will be determined as follows: 0.5 ml aliquotswill be mixed at room temperature with 20 μl 10M sodium hydroxide. Anammonium specific electrode (such as one obtainable from Orion ResearchInc., Cambridge, Mass.) will be inserted into the mixture and thevoltage generated by ammonia will be determined. Solutions with knownconcentrations of ammonium chloride will be used for calibration of theammonia specific electrode.

EXAMPLE 3

The activity of the compounds of this invention to prevent or reduce theaccumulation of β-amyloid plaques and thus the usefulness in thetreatment of senile dementia of the Alzheimer's type and otherconditions known to be associated with the formation of β-amyloid plaquesuch as Down's syndrome can be demonstrated by various in vitro and invivo models of β-amyloid plaque formation. For example the ability ofthe compounds of this invention to prevent or reduce the accumulation ofβ-amyloid depositions can be demonstrated by several cellular and cellfree in vitro methods described as Assay's 1-3 as follows. These assaysmake use of the fact that native β-APP is expressed by cells and isprocessed to produce 11-12 KDa C-terminal fragments and β-amyloid. Theendogenous level of β-APP expression can be enhanced if desired bytransfecting β-APP cDNA sequences, e.g., β-APP (751) into the cellsusing standard methodology.

IN VITRO ASSAYS

Assay #1: immunoprecipitation

Cells: CHO-K1 (Chinese Hamster Ovary; ATCC origin) cell line stablytransfected to express large amounts of βAPP-695, and referred to as"CP-6-36" are used for screening of β-APP depositions. Other mammaliancultured cell lines can also be used and have been used. For example,the human neuronal cell line SK-N-ML (ATCC origin) gives good resultsunder the same assay conditions. Transfection with βAPP-695 is not arequisite of 8A4 production; it merely enhances the βA4 signal. Inpreparation for an experiment, CP-6-36 cells are seeded at low densityin 10 cm dishes and grown for two to four days to a confluent monolayer(˜1.5×10⁷ cells per dish) in a 37° C. /5 CO₂ incubator; growth mediaconsists of DMEM 21/Coon's F12 (1:1)+10% FBS (fetal bovine serum) +50U/mL penicillin and 50 μg/mL streptomycin.

Treatment: All compounds are initially screened on CP-6-36 cells at adose of 200 μM. Prior to testing, a 20 mM stock of each compound to betested is prepared using cell culture grade DMSO as a solvent. Each 20mM stock compound is then diluted 100-fold into serum free EMEM mediadeficient in the amino acids cysteine and methionine ("Cys-/Met- EMEM"),giving a 200 μM final concentration of compound in the media. To beginthe experiment the cells are "starved" for cysteine and methionine bywashing the cell monolayers 3 times with 3 mL/dish of Cys-/Met- EMEM,then incubating (37° C./5% CO₂) with 3 mL/dish of the same media for 15minutes. This media is aspirated from the dishes, then media containingthe compounds at 200 μM is added at 3 mL/dish. These plates and a"control" dish (3 mL/dish Cyst-/Met- EMEM containing 1% DMSO and nocompound) are incubated as above for 15 minutes. This media isaspirated, then to each dish an additional 3 mL of the media from theprevious step now containing ³⁵ S-Trans label (³⁵ -S labeled cysteineand methionine) at ˜150 μCi/mL is added. The cells are incubated asabove for 4 hours.

Harvest: At the end of the 4 hour labeling period, the cells areobserved under the microscope for overall appearance and to check forgross toxicity effects of the compounds, after which the dishes of cellsare placed on ice. The conditioned media from each dish is transferredto 15 mL conical screw-cap tubes, centrifuged at 2000 rpm for 10 minutesand transferred to a set of similar tubes, leaving behind any pelletedcells. The labeled cell monolayers are washed three times with 2 mL/dishphosphate-buffered saline (PBS), then 1 mL of a buffer which promotescell lysis (5% Triton X-114; 20 mM Tris, pH 7.5; 300 mM NaCl; proteaseinhibitors) is added to each dish, followed by a 10 minute incubation onice. The cell lysates are scraped from the dishes and transferred to 1.5mL microfuges tubes. The lysates are then sonicated for 4 minutes onice, spun at high speed in a microfuge for 10 minutes, then transferredto 15 mL conical screw-cap tubes, leaving behind the pellet of celldebris.

Immunoprecipitation: In preparation for immunoprecipitation, the lysatesharvested above are diluted in 5 mL of 1×RIPA buffer (10 mM Tris, pH8.0; 150 mM NaCl; 0.125% NAN₃ ; 1% Triton X-100; 1% deoxycholate; 0.1SDS); the conditioned media samples are immunoprecipitated withoutdilution. Both conditioned media and lysates are first precleared byadding 5 μL of normal rabbit serum to each sample, rocking 10 minutes atroom temperature, followed by the addition of 100 μL 10% proteinA-Sepharose (PAS) in RIPA buffer, and rocking at room temperature for1.5 hours. The samples are then centrifuged at 3000 rpm, and thesupernatants are transferred to new 15 mL tubes. The precleared lysatesare then immunoprecipitated by adding 30 μL of an antibody whichrecognizes the carboxyl terminus of βAPP to each tube, rocking for 10minutes at room temperature, followed by the addition of 100 μL of 10%PAS and rocking at room temperature for 1.5 hours. The preclearedconditioned media samples are immunoprecipitated identically, however 45μL of an antibody which recognizes βA4 is used instead of the carboxylterminal directed antibody. All samples are then centrifuged for 1minute at 3000 rpm to pellet the PAS-antibody complexes, and theresulting pellets are washed extensively; 4 times with a high saltbuffer (50 mM Tris, pH 7.5; 500 mM NaCl; 5 mM EDTA; 0.5% Nonidet P-40),3 times with a low salt buffer (50 mM Tris, pH 7.5; 150 mM NaCl; 5 mMEDTA; 0.5 Nonidet P-40), and 2 times with 10 mM Tris buffer, pH 7.5.

Gel electrophoresis: The washed pellets are boiled for 5 minutes in 50μL of 2×Laemmli gel loading buffer. These samples as well as molecularweight markers are loaded onto a 16.5% SDS-polyacrylamide gel withTris/Tricine reservoir buffers. The gel is run at 90 V for ˜18-20 hours,fixed in 20% methanol/20% acetic acid, and dried onto filter paper at65° C. for 2 hours. Autoradiography is used to visualize the results.

Analysis: Results are obtained by analysis of the autoradio-gram. Apositive acting compound is one which inhibits the 4 kDa μA4 proteinband relative to the control sample, and which increases levels of the9-12 kDa C-terminal protein bands relative to the control sample.Quantitation of inhibition of βA4 or increase of C-terminal bands can bemade by densitometric scanning of the bands, normalized to controlbands. A negative acting compound is one which shows no change in theyield of 4 kDa βA4 or 9-12 kDa C-terminal protein bands, relative to thebands from the control sample.

Additional testing: If a compound is to be found to be active (i.e.,substantial inhibition of 4 kDa βA4 formation with concomitant increasein C-terminal fragments, by gel analysis), then a dose responseexperiment is performed to determine the lowest dose of compoundnecessary to elicit above effects. The dose range typically used is12.5-300 μM, and with the exception of these dose changes, theexperiment is done identically as described above. If a compound isfound to be only slightly active or not active at all, the experiment isrepeated using a higher dose, typically 400 μM. If a compound is foundto be toxic (i.e., cells appear unhealthy by observation under themicroscope, or lysates appear to not have been labeled well after gelanalysis), then the compound is tested again at lower doses, forexample: 25, 50 and 100 μM, to determine the effect of the compound at anon-toxic dose.

Assay #2: Radioimmunoassay

Preparation and Sepak concentration of media for the RIA: Culturedmammalian cells such as Chinese hamster ovary (CHO) cells or humanneuronal SK-N-ML cells produce β-amyloid and secrete this peptide intothe culture medium. If cells are treated with potential inhibitors ofβ-amyloid formation, no soluble β-amyloid would be found in the mediumof the treated cells. As with Assay #1, varying doses of inhibitorycompounds can be tested beginning with 200 μM. For CHO cells, both wildtype and β-APP695 transfected, 10 cm plates are incubated in 2 mL EMEM(serum free) for 4 to 6 hours at 37° C. in the presence or absence ofinhibitory compounds to be evaluated. The medium is removed andcentrifuged for 10 minutes at 1500 rpm (Sorvall RT6000B) to remove anycells/debris. The medium is either used immediately or stored at -20° C.

The Sepak C18 step is performed to remove salts and other unwantedcontaminants and to concentrate the β-amyloid peptides. Medium sample (2ml) is passed through a C18 Sepak cartridge and the cartridge is washedin 2 ml 5% CH₃ CN in 0.1% TFA. The runthrough and the 5% CH₃ CN wash arediscarded. The cartridge is eluted with 2 mL 25% CH₃ CN in 0.1% TFAfollowed by 2 mL elution in 50% CH₃ CN in 0.1% TFA. Both elutions arecollected and dried in the speedvac and taken up in 125 μL to 250 μL of10% isopropanol in water for assaying in the RIA. The 25% CH₃ CNfraction contains most of the phenol red from the media but no β-amyloidpeptide. The 50% CH₃ CN fraction contains the β-amyloid peptides.

Preparation and HPLC purification of ¹²⁵ I labeled β-amyloid 1-40:Synthetic β-amyloid 1-40 (10 μg) is labeled with ¹²⁵ I (1 mCi) by theChloramine T method. The reaction is carried out at room temperature. Inan Eppendorf tube, 10 μL of ¹²⁵ I (1 mCi in NAOH solution) is added to10 μL of β-amyloid 1-40 (1 mg/mL in 20% Isopropanol) and 80 μL 0.1MNaPhosphate, pH 7.4 and mixed. The reaction is initiated by adding 30 μLChloramine-T (1 mg/mL, in 0.1M NaPhosphate, pH 7.4) mixing andincubating 1 minute. The reaction is stopped by adding 150 μLNaMetabisulfite (2mg/mL, 0.1M NaPhosphate, pH 7.4).

The reaction mixture (280 μL) is diluted with equal volume of water andrun on a Sepak C18 cartridge to separate the labeled peptide. The Sepakis washed twice in 5% CH₃ CN (1 mL each) and eluted three times in 50%CH₃ CN (1 mL each) and washed again twice in 95% CH₃ CN (1 mL each).Almost all of the labeled peptide elutes in the first 50% CH₃ CNelution. This elution is stored at -70° C. and purified by HPLC asneeded for the RIA.

The labeled peptide is purified by reverse phase HPLC on a C8 cartridge(4.6 mm×3 cm, Brownlee). The column is run in a linear gradient from 5%to 45% CH₃ CN in 0.1% TFA in 30 minutes at a flow rate of 0.5 ml/min.Fractions (0.5 mL) are collected and counted. The peak fractioncontaining the labeled peptide is stored at -20° C. and used within 3days in the RIA.

Radioimmunoassay: The buffers used in the RIA are 1) RIA buffer: 0.1MNaPhosphate, pH 7.4 containing 0.1% BSA and 0.1% Triton-X-100.2) Samplebuffer: 10% Isopropanol in water. 3) Tracer buffer: 0.2M NaPhosphate, pH7.4 containing 0.1% BSA in 0.1% Triton-X-100.The β-amyloid specificantibodies are used at dilutions where approximately 30% of the labeledpeptide is bound in the absence of competing ligand. The dilutions ofthe antibodies are prepared in RIA buffer. The antibodies used in theRIA include three different sera raised to human β-amyloid 1-40synthetic peptide (BA#1, BA#2, and 6514). BA#1 is used at final dilutionof 1/900, BA#2 at 1/1600 and 6514 at 1/2500. The HPLC purified labeledpeptide is diluted in tracer buffer to give between 7000 and 9000 cpm in50 μL. Total displacement is done in the presence of high concentration(2.5 μM) of β-amyloid 1-40. The β-amyloid 1-40 standards are prepared insample buffer. The assay volume is 200 μL. Components are added in thefollowing order:

100 μL Ab in RIA buffer

50 μL Unknown sample or standard or TD in sample buffer

50 μL Labeled peptide (7000-9000 cpm in tracer buffer)

The samples are mixed and incubated overnight at 4° C. To separate thebound counts from the free counts, the assay is terminated withpolyethylene glycol (PEG). To each assay tube, 50 μL of normal rabbitserum is added, followed by 800 μL of PEG (MW6000-8000, 15.8% in RIAbuffer). The samples are incubated for 10 minutes at 4° C. andcentrifuged 3200 rpm, 20 minutes (Sorvall, RT600B). The supernatant isaspirated and the pellets are counted in the gamma counter.

Analysis: Results from antibody binding are interpreted based ondisplacement of the labeled β-amyloid tracer. A positive result is onein which no displacement of tracer is observed, i.e., medium does notcontain secreted β-amyloid indicating the compound tested is effectivein inhibiting β-amyloid production. A negative result is one in whichdisplacement of tracer for antibody binding is seen and equivalent tountreated control cells.

An enzyme linked immunosandwich assay (ELISA) can also be employed toidentify active compounds. Cultured mammalian cells (such as CHO CP-6 orSK-N-MC) producing β-amyloid protein are prepared and treated withcompounds as described for Assay #1 except that radiolabelling of cellprotein is eliminated. Conditioned media from treated cell cultures isharvested and clarified of cellular debris by low-speed centrifugation.The conditioned media is then assayed in a 96 well ELISA formatutilizing β-amyloid-specific antibodies. One β-amyloid antibody servesas the capture reagent for the β-amyloid present in the media samples,the second β-amyloid-specific antibody which recognizes a differentepitope on the β-amyloid protein serves as a component of the detectorcomplex. The second β-amyloid antibody is conjugated with biotin whichcan be detected by strept-avidin. A third antibody which is coupled tohorseradish peroxidase is used to detect the β-amyloid:antibody;strept-avidin complex. Addition of o-phenylenediamine substrate plus H₂O₂ and citrate phosphate pH 5 allows for peroxidase activity which isquantitated by reading the colorimetric change in the mixture at OD⁴⁹⁰nm. Typically, serial three-fold dilutions of each medium sample is madein the 96 well plate in addition to a standard, synthetic β-amyloid 1-40protein. A positive result is one in which little or no reactivity,i.e., adsorbance at OD⁴⁹⁰ nm, is obtained indicated the absence ofβ-amyloid protein in the medium sample as a result of inhibition by thecompound tested. Partially active inhibitors would give some but notequivalent absorbance at OD^(490nm) to a control medium sample fromuntreated cells. Precise quantitation can be achieved by comparingsample values to the standard.

IN VIVO ASSAYS

The activity of the compounds of this invention to prevent or reduce theaccumulation of β-amyloid plaques can be demonstrated in an transgenicmouse model of β-amyloid plaque accumulation and in a dog model usingdogs with a natural, genetic predisposition to the formation ofβ-amyloid plaque. Transgenic mice which overexpress human β-APP (751) orβ-APP (770) in neuronal cells and display histopathology associated withAlzheimer's disease are described, for example, in PCT/US91/04447. Insuch animal models, the reduction of histopathology and/or symptomsassociated with β-amyloid depositions such as memory loss, can be usedto demonstrate the ability of the compounds to treat the therapeuticconditions resulting from β-amyloid plaque formation such as Alzheimer'sDisease and the memory impairment associated with Down's syndrome.

Since the histopathology in the transgenic mice is more frequent withincreased age of the animal, 2 month old mice would be desirable. The 2month animals would have minimal pathology which would increase withtime in the absence of inhibitory drug. All animals in the experimentwould be from a single pure bred pedigree. One group of mice (n=12)would receive vehicle only; a second group (n=12) would receive a lowdose of drug; a third group (n=12) a moderate dose; and a fourth group(n=12) a high dose. Dosage would be determined from the above assaystaking into account body weight, compound half-life, etc. Ideally, micewould be treated for several months. Delivery of the compound could beby injection, oral route, an implant with timed release, etc., asdictated by the compound profile. Evaluation of treatment would be madeusing immuno-histochemistry to determine the frequency of β-amyloidimmunoreactive deposits in coronal midline sections of brain scored byan investigator blinded from the experimental treatment. Another markerof pathology, Alz50 immunoreactivity, would also be scored for frequencyof occurrence using the same number of brain tissue sections from allmice in the study. A positive result of drug action would be the absenceor reduced frequency of both pathological markers. A physiologicaland/or behavioral correlate unique to the β-amyloid transgenic mice canalso be used to demonstrate drug action.

Some canine races have been reported to have β-amyloid accumulations(Giaccone et al., Neuroscience Letters Vol.114, pp 178-183 (1990)). Agednon-human primates display β-amyloid pathology, as well as memoryimpairments (Cork et al., American Journal of Pathology, Vol .137, pp1383-1392 (1990)); Podlisny et al., American Journal of PathologyVol.138, pp 1423-1425 (1991)). Tests with canines and non-human primateswould most likely follow a somewhat different experimental design withdrug application time being longer.

EXAMPLE 4

In order to test the efficacy of treatment using the compounds of thepresent invention on the cognitive and noncognitive behavioraldysfunctions in DAT patients, the ADAS (Alzheimer's Disease AssessmentScale) can be used according to the method of W. G. Rosen, et al., Am.J. Psychiatry 141: 1356-1364 (1984), incorporated herein by reference.Cognitive functioning can be assessed on 17 items. These include memoryfunctions, language functions and ideational praxis task andconstructional praxis. Noncognitive behaviors will be rated on 23 itemswhich include mood state (depression, anxiety), vegetative symptoms,socialization skills, cooperation, initiative for activities of dailyliving, psychotic symptoms, motor activity, agitation, concentration andnocturnal confusion.

EXAMPLE 5

This example describes procedures useful in determining other agentsuseful in combination therapy with the compounds of the presentinvention and for use alone.

Materials and Methods:

Chemicals: Usual laboratory chemicals were obtained from Baker Chemical(Derenter, The Netherlands) or Merck (Darmstadt, Germany). L-Carnitine,L-acetylcarnitine, N-acetyl-L-glutamate and the amino acids L-ornithine,L-arginine and L-citrulline were from Sigma Chemical Co. (St. Louis,Mo.). 5-Fluoromethylornithine.2HCl .H₂ O (5FMOrn) is a compound of thepresent invention.[(+)-5-Methyl-10,11-dihydro-5H-dibenzo[a,d]cycloheptene-5,10-imine) wasfrom Bioblock Scientific (Illkirch, France).(R)-4-oxo-5-phosphono-norvaline (Whitten et al., J. Med. Chem. (1990)11:2961-2963, is a product of Marion Merrell Dow Inc.

Laboratory animals: Female CDl mice (Charles River, St.Aubin-les-Elbeuf, France) were kept in groups of 10 under standardizedconditions (water and standard rodent chow ad libitum, 22° C., 60%relative humidity, 12 hours light, 12 hours dark cycle).

Drug regimen and intoxication with ammonium acetate: Mice are pretreatedby i.p. administration of 5-fluoromethylornithine, usually 16 hoursbefore the i.p. injection of 13 or 15 mmol.kg⁻¹ ammonium acetate (1.00g, respectively 1.15 g in 10 ml of water; 0.1 ml per 10 g body weight).Amino acids and the other compounds are given subcutaneously (s.c.)usually 1 hour before intoxication with ammonium acetate. (Treatmentsdifferent from these are mentioned in the legends of Tables.)

After intoxication with ammonium acetate the appearance of clonicseizures, the loss of the righting reflex, coma, and tonic hind limbextensions are recorded. Survivors exhibit normal behavior 2 hours afterintoxication.

Tissue preparation: Mice are decapitated and brains rapidly (<10seconds) isolated, frozen in liquid nitrogen, and stored at -80° C.until analysis. For the determination of ammonia, glutamine andglutamate the frozen brains are homogenized in 10 volumes of ice-cold0.2M perchloric acid. After 1 hour at 2° C. the homogenates werecentrifuged, and the supernatants submitted to the assay procedures onthe same day, in order to minimize hydrolysis of glutamine or of otherhydrolytically releasable forms of ammonia.

Amino Acid and ammonia: For the determination of glutamine and glutamatealiquots of the perchloric acid, brain extracts are separated by usingthe isocratic elution mode of a previously published reversed-phase HPLCmethod (Seiler and Knodgen, 1985). For ammonia determinations, anammonia selective electrode (Orion Research Inc., Cambridge, U.S.A.) isused. Standard solutions of ammonium chloride are prepared for theestablishment of a calibration curve for each series of samples. (Fordetails, see Seiler et al., 1992.)

                                      TABLE 1                                     __________________________________________________________________________    Protection of mice against intoxication with 13 mmol · kg.sup.-1     (i.p.) ammonium acetate by                                                    amino acids and related compounds alone and in combination with 5 μmol     · kg.sup.-1 5-fluoro-                                                methylornithine (5FMorn)                                                                                   Percent                                                               with loss of                                                                          animals with tonic                                                                     Percent                                 Drug        Dose mmol · kg.sup.-1                                                         righting reflex                                                                       seizure  survivors                               __________________________________________________________________________    A. Single drug treatment                                                      Vehicle (3% NaHCO.sub.3)                                                                  --       100     100       0                                      L-Ornithine 10       90      20       90                                      L-Arginine  10       80      10       90                                      L-Citrulline                                                                              5        70      10       100                                                 3        70      10       90                                                  0.75     90      70       40                                      N-Acetyl-L-glutamate                                                                      5        100     90       10                                      L-Carnitine 15       90      90       10                                      L-Acetylcarnitine                                                                         15       100     70       30                                      B. Combined treatment with 5 μmol · kg.sup.-1 5FMorn              Physiol. saline                                                                           --       60      60        40*                                    L-Citrulline                                                                              0.75     90      30        80*                                    N-Acetyl-L-glutamate                                                                      5        90      40       20                                      L-Carnitine 15       100     50        60*                                    L-Acetylcarnitine                                                                         15       90      40       100*                                    __________________________________________________________________________

Groups of 10 CDl mice (female, weighing 22±3 g received either 5μmol.kg⁻¹ 5FMOrn (i.p.) or physiol. saline. 15 hours later theabove-mentioned drugs dissolved in 3% NaHCO₃ were administeredsubcutaneously, and another hour later 13 mmol.kg⁻¹ ammonium acetate (inwater) was injected i.p.

The asterisk (*) indicates a statistically significant difference(p≦0.05) between single drug and combination treatment; (Non-parametricstatistics (Siegel, 1956).)

                                      TABLE 2                                     __________________________________________________________________________    Intoxication of mice with 15 mmol · kg.sup.-1 (i.p.) ammononium      acetate. Effects of                                                           pretreatment with 0.1 mmol · kg.sup.-1 5-fluoromethylornithine       (5FMorn) and other compounds                                                  known to antagonize acute ammonia intoxication                                                   Percent animals   with loss of                                                with tonic seizure                                                                     with tonic seizure                                                                     righting reflex                                             prior to the loss                                                                      after the loss of                                                                      without tonic                            Treatment          of righting reflex                                                                     righting reflex                                                                        seizure survivors                        __________________________________________________________________________    None               100      0        0       0                                5FMorn             35  (20-50).sup.a                                                                      63  (50-80).sup.a                                                                      2  (0-10).sup.a                                                                       2  (0-10).sup.a                  L-Citrulline (5 mmol · kg.sup.-1)                                                       70       30       0       0                                5FMorn + L-Citrulline                                                                            30       70       0       0                                L-Ornithine (10 mmol · kg.sup.-1)                                                       0        100      0       10                               5FMorn + L-Ornithine                                                                             0        80       20      20                               L-Arginine (10 mmol · kg.sup.-1)                                                        40       60       0       0                                5FMorn + L-Arginine                                                                              30       70       0       0                                L-Carnitine (15 mmol · kg.sup.-1)                                                       100      0        0       0                                5FMorn + L-Carnitine                                                                             0        60       40      60*                              L-Acetylcarnitine (15 mmol · kg.sup.-1)                                                 0        100      0       0                                5FMorn + L-Acetylcarnitine                                                                       0        50       50      60*                              N-Acetyl-L-glutamate (5 mmol · kg.sup.-1)                                               30       70       0       0                                5FMOrn + N-Acetyl-L-glutamate                                                                    30       70       0       0                                __________________________________________________________________________     .sup.a Mean value of four independent experiments; range in parentheses. 

Each group consisted of 10 female CDl mice (weighing 20±2 g).Pretreatment with 0.1 mmol.kg⁻¹ (i.p.) 5FMOrn 16 hours before 15mmol.kg⁻¹ ammonium acetate administration (i.p.). Amino acids andrelated compounds were given s.c. 1 hour before ammonium acetate.

The asterisk (*) indicates a statistically significant (p=0.05)difference between groups treated with a single drug and the combinationwith 5FMOrn; non-parametric statistics (Siegel, 1956).

Ammonium acetate, 5FMOrn and ornithine, arginine, and citrulline weredissolved in water; carnitine, acetylcarnitine and N-acetylglutamate in2% NaHCO₃ (0.1 ml per 10 g body weight).

                                      TABLE 3                                     __________________________________________________________________________    Ammonia, glutamine and glutamate in the brain of mice, 10 min after           administration of                                                             ammonium acetate. Effect of pretreatment with 5-fluoromethylornithine         (5FMOrn) and                                                                  other compounds known to antagonize acute ammonia intoxication                                        Glutamine                                                                            Glutamate                                                                            Glutamine                               Treatment        Ammonia                                                                              μmol                                                                              per g  Glutamate                               __________________________________________________________________________    None.sup.(a)     0.9 ± 0.1                                                                         5.9 ± 0.4                                                                         12.6 ± 0.3                                                                        0.47 ± 0.03                          Ammonium acetate 2.7 ± 0.5                                                                         10.3 ± 0.8                                                                        10.5 ± 0.4                                                                        0.98 ± 0.09                          5FMOrn (0.1 mmol · kg.sup.-1)                                                         1.5 ± 0.2*                                                                        8.9 ± 0.7                                                                         10.8 ± 0.5                                                                        0.82 ± 0.05                          Ornithine (10 mmol · kg.sup.-1)                                                       1.5 ± 0.4*                                                                        8.8 ± 0.5*                                                                        11.5 ± 0.5                                                                         0.77 ± 0.03*                        Ornithine + 5FMOrn                                                                             1.3 ± 0.2*                                                                        8.7 ± 0.2*                                                                        11.3 ± 0.2                                                                        0.77 ± 0.03                          Arginine (10 mmol · kg.sup.-1)                                                        1.5 ± 0.2*                                                                        8.9 ± 0.3*                                                                        10.2 ± 1.0                                                                        0.89 ± 0.07                          Arginine + 5FMOrn                                                                              1.8 ± 0.3*                                                                        8.4 ± 0.9*                                                                        10.2 ± 0.9                                                                        0.82 ± 0.04                          Citrulline (5 mmol · kg.sup.-1)                                                       1.6 ± 0.5*                                                                        7.3 ± 0.4*                                                                        11.9 ± 0.5                                                                        0.61 ± 0.04                          Citrulline + 5FMOrn                                                                            1.3 ± 0.2*                                                                        7.0 ± 0.4*                                                                        11.6 ± 0.4                                                                         0.60 ± 0.02*                        N-Acetylglutamate (5 mmol · kg.sup.-1)                                                1.8 ± 0.5*                                                                        9.2 ± 0.9                                                                          11.3 ± 0.5*                                                                      0.81 ± 0.08                          N-Acetylglutamate + 5FMOrn                                                                      1.2 ± 0.1**                                                                       8.2 ± 0.3**                                                                      11.9 ± 0.4                                                                         0.69 ± 0.04**                       L-Carnitine (15 mmol · kg.sup.-1)                                                     1.7 ± 0.5*                                                                        8.8 ± 0.4*                                                                         11.4 ± 0.3*                                                                      0.77 ± 0.06                          L-Carnitine + 5FMOrn                                                                            1.2 ± 0.2**                                                                       7.8 ± 0.7**                                                                      11.9 ± 0.3                                                                        0.65 ± 0.04                          L-Acetylcarnitine (15 mmol · kg.sup.-1)                                               2.5 ± 0.3                                                                         8.9 ± 0.6                                                                          11.4 ± 0.4*                                                                      0.78 ± 0.04                          L-Acetylcarnitine + 5FMOrn                                                                      1.4 ± 0.3**                                                                       7.8 ± 0.4**                                                                       11.5 ± 0.8*                                                                       0.67 ± 0.03**                       __________________________________________________________________________     5-Fluoromethylornithine (0.1 mmol · kg.sup.-1) was given i.p. 16     hours before 8 mmol · kg ammonium acetate (i.p.). Amino acids an     related compounds were administered subcutaneously 1 hour before ammonium     acetate. The animals were killed by decapitation 10 minutes after             administration of ammonium acetate.                                           .sup.(a) With the exception of these animals, all other groups received 8     mmol · kg.sup.-1 ammonium acetate.                                   *Statistically significant difference (p ≦ 0.05) between ammonium      acetate treated "controls" and groups receiving pretreatment with a drug.     **Statistically significant difference (p ≦ 0.05) between groups       receiving a single drug, and the drug combination with 5FMOrn (Student's      ttest).                                                                  

For pharmacological end-use applications, the compounds of Formula I arepreferentially administered in the form of their pharmaceuticallyacceptable acid addition salts. Of course, the effective dosage of thecompounds will vary according to the individual potency of each compoundemployed, the severity and nature of the disease being treated and theparticular subject being treated. In general, effective results can beachieved by administering a compound at a dosage of about 0.01 mg toabout 20 mg per kilogram of body weight per day of the compounds offormula I administered systemically. Therapy should be initiated atlower dosages. The dosage thereafter may be administered orally in soliddosage forms, e.g., capsules, tablets, or powders, or in liquid forms,e.g., solutions or suspensions. The compounds may also be injectedparenterally in the form of sterile solutions or suspensions. Forcombination therapy, therapeutic agents administered concurrently orconsecutively to administration with compounds of Formula I areadministered preferably in a dosage of about 0.1 mg to about 100 mg perkilogram of body weight per day.

In practicing the method of this invention, the compounds of formula Iand/or the therapeutic agents in combination therapy are preferablyincorporated in a composition comprising a pharmaceutical carrier andfrom about 5 to about 90 percent by weight of a compound of theinvention or a pharmaceutically acceptable salt thereof. The term"pharmaceutical carrier" refers to known pharmaceutical excipientsuseful in formulating pharmaceutically active compounds for internaladministration to animals, and which are substantially non-toxic andnon-sensitizing under conditions of use. The compositions can beprepared by known techniques for the preparation of tablets, capsules,elixirs, syrups, emulsions, dispersions and wettable and effervescentpowders, and can contain suitable excipients known to be useful in thepreparation of the particular type of composition desired.

The preferred route of administration is oral administration. For oraladministration the active compounds can be formulated into solid orliquid preparations such as capsules, pills, tablets, troches, lozenges,melts, powders, solutions, suspensions, or emulsions. The solid unitdosage forms can be a capsule which can be of the ordinary hard- orsoft-shelled gelatin type containing, for example, surfactants,lubricants, and inert fillers such as lactose, sucrose, calciumphosphate, and cornstarch. In another embodiment the compounds of thisinvention can be tableted with conventional tablet bases such aslactose, sucrose, and cornstarch in combination with binders such asacacia, cornstarch, or gelatin, disintegrating agents intended to assistthe break-up and dissolution of the tablet following administration suchas potato starch, alginic acid, corn starch, and guar gum, lubricantsintended to improve the flow of tablet granulations and to prevent theadhesion of tablet material to the surfaces of the tablet dies andpunches, for example, talc, stearic acid, or magnesium, calcium, or zincstearate, dyes, coloring agents, and flavoring agents intended toenhance the aesthetic qualities of the tablets and make them moreacceptable to the patient. Suitable excipients for use in oral liquiddosage forms include diluents such as water and alcohols, for example,ethanol, benzyl alcohol, and the polyethylene alcohols, either with orwithout the addition of a pharmaceutically acceptable surfactant,suspending agent, or emulsifying agent.

The active compounds of this invention may also be administeredparenterally, that is, subcutaneously, intravenously, intramuscularly,or interperitoneally, as injectable dosages of the compound in aphysiologically acceptable diluent with a pharmaceutical carrier whichcan be a sterile liquid or mixture of liquids such as water, saline,aqueous dextrose and related sugar solutions, an alcohol such asethanol, isopropanol, or hexadecyl alcohol, glycols such as propyleneglycol or polyethylene glycol, glycerol ketals such as2,2-dimethyl-l,3-dioxolane-4-methanol, ethers such as polyethyleneglycol 400, an oil, a fatty acid, a fatty acid ester or glyceride, or anacetylated fatty acid glyceride with or without the addition of apharmaceutically acceptable surfactant such as a soap or a detergent,suspending agent such as pectin, carbomers, methylcellulose,hydroxypropylmethylcellulose, or carboxymethylcellulose, or emulsifyingagent and other pharmaceutically acceptable adjuvants. Illustrative ofoils which can be used in the parenteral formulations of this inventionare those of petroleum, animal, vegetable, or synthetic origin, forexample, peanut oil, soybean oil, sesame oil, cottonseed oil, corn oil,olive oil, petrolatum, and mineral oil. Suitable fatty acids includeoleic acid, stearic acid, and isostearic acid. Suitable fatty acidesters are, for example, ethyl oleate and isopropyl myristate. Suitablesoaps include fatty alkali metal, ammonium, and triethanolamine saltsand suitable detergents include cationic detergents, for example,dimethyl dialkyl ammonium halides, alkyl pyridinium halides; anionicdetergents, for example, alkyl, aryl, and olefin sulfonates, alkyl,olefin, ether, and monoglyceride sulfates, and sulfosuccinates; nonionicdetergents, for example, fatty amine oxides, fatty acid alkanolamides,and polyoxyethylene-polypropylene copolymers; and amphoteric detergents,for example, alkyl beta-aminopropionates, and 2-alkylimidazolinequarternary ammonium salts, as well as mixtures. The parenteralcompositions of this invention will typically contain from about 0.5 toabout 25% by weight of the formula I compound in solution. Preservativesand buffers may also be used advantageously. In order to minimize oreliminate irritation at the site of injection, such compositions maycontain a non-ionic surfactant having a hydrophile-lipophile balance(HLB) of from about 12 to about 17. The quantity of surfactant in suchformulations ranges from about 5 to about 15% by weight. The surfactantcan be a single component having the above HLB or can be a mixture oftwo or more components having the desired HLB. Illustrative ofsurfactants used in parenteral formulations are the class ofpolyethylene sorbitan fatty acid esters, for example, sorbitanmonooleate and the high molecular weight adducts of ethylene oxide witha hydrophobic base, formed by the condensation of propylene oxide withpropylene glycol.

The compounds of the present invention can also be administeredtopically. This can be accomplished by simply preparing a solution ofthe compound to be administered, preferably using a solvent known topromote transdermal absorption such as ethanol or dimethyl sulfoxide(DMSO) with or without other excipients. Preferably topicaladministration will be accomplished using a patch either of thereservoir and porous membrane type or of a solid matrix variety.

Some suitable transdermal devices are described in U.S. Pat. Nos.3,742,951, 3,797,494, 3,996,934, and 4,031,894. These devices generallycontain a backing member which defines one of its face surfaces, anactive agent permeable adhesive layer defining the other face surfaceand at least one reservoir containing the active agent interposedbetween the face surfaces. Alternatively, the active agent may becontained in a plurality of microcapsules distributed throughout thepermeable adhesive layer. In either case, the active agent is deliveredcontinuously from the reservoir or microcapsules through a membrane intothe active agent permeable adhesive, which is in contact with the skinor mucosa of the recipient. If the active agent is absorbed through theskin, a controlled and predetermined flow of the active agent isadministered to the recipient. In the case of microcapsules, theencapsulating agent may also function as the membrane.

In another device for transdermally administering the compounds inaccordance with the present invention, the pharmaceutically activecompound is contained in a matrix from which it is delivered in thedesired gradual, constant and controlled rate. The matrix is permeableto the release of the compound through diffusion or microporous flow.The release is rate controlling. Such a system, which requires nomembrane is described in U.S. Pat. No. 3,921,636. At least two types ofrelease are possible in these systems. Release by diffusion occurs whenthe matrix is non-porous. The pharmaceutically effective compounddissolves in and diffuses through the matrix itself. Release bymicroporous flow occurs when the pharmaceutically effective compound istransported through a liquid phase in the pores of the matrix.

What is claimed is:
 1. A process of making(2S,5S)-5-fluoromethylornithine comprising:adding a sufficient amount ofwet penicillin acylase to a solution of (2R,5R) and(2S,5S)-6-fluoromethyl-3-(phenylacetyl)-amino-2-piperidone; recovering((2S,5S)-6-fluoromethyl-3-amino-2-piperidone from the solution; andadding a sufficient amount of an acid to produce(2S,5S)-5-fluoromethylornithine.